Earlier we reported that cyclic GMP is involved in the stimulus-secretion coupling in the exocrine pancreas and calcium appears to be involved in this action. In this study, we have examined the possible role of cacium and other divalent ions in the activation of guanylate cyclase of pancreas. Guanylate cyclase in pancreas is present in both soluble and membrane bound forms and shows an absolute requirement for Mn ions. The substrate for the guanylate reaction is MnGTP and has a site where free divalent ions intact increasing the enzyme activity. Calcium in very low concentrations (10 micron M) increases the soluble guanylate cyclase activity in pancreas by 50% when assayed at low substrate concentrations (MnGTP, 100 micrn M); maximal activation is attained at 500 micon M Ca ions. Other divalent ions (Ba ions, Co ions, Zn ions, Fe ions) do not activate the enzyme. These ions inhibit the guanylate cyclase in both the soluble and membrane bound fractions. Acetylcholine, carbamyl choline, cerulenin, CCK-OP, which induce marked increase in cyclic GMP in pancreas, do not activate the enzyme in vitro. All these agents, however, cause marked increase in calcium efflux from pancreas. It is possible that calcium from an intracellular pool may be involved in the activation of guanylate cyclase and hence an increase in cyclic GMP in pancreas.